RESUMO
KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Histonas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Transdução de Sinais , Linhagem Celular TumoralRESUMO
Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.
Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming 'induced pluripotent stem cells'; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the 'precursor' cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow 'induced myoblasts' from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.
Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Distrofia Muscular Facioescapuloumeral/terapia , Mioblastos/transplante , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Desenvolvimento Muscular , Distrofia Muscular Facioescapuloumeral/patologia , Fator de Transcrição PAX3/metabolismo , Recuperação de Função Fisiológica , RegeneraçãoRESUMO
Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.
Assuntos
Hiperaldosteronismo/genética , Hipertensão/genética , Receptor alfa de Ácido Retinoico/genética , Fator A de Crescimento do Endotélio Vascular/genética , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/patologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Proliferação de Células/genética , Matriz Extracelular/genética , Humanos , Hiperaldosteronismo/patologia , Hipertensão/patologia , Camundongos , Camundongos Knockout , Mutação/genética , Via de Sinalização Wnt/genéticaRESUMO
Macrophage mineralocorticoid receptor (MR) signaling is an important mediator of cardiac tissue inflammation and fibrosis. The goal of the present study was to determine the cellular mechanisms of MR signaling in macrophages that promote cardiac tissue injury and remodeling. We sought to identify specific markers of MR signaling in isolated tissue macrophages (cardiac, aortic) vs splenic mononuclear cells from wild-type and myeloid MR-null mice given vehicle/salt or deoxycorticosterone (DOC)/salt for 8 weeks. Cardiac tissue fibrosis in response to 8 weeks of DOC/salt treatment was found in the hearts from wild-type but not myeloid MR-null mice. This was associated with an increased expression of the profibrotic markers TGF-ß1 and matrix metalloproteinase-12 and type 1 inflammatory markers TNFα and chemokine (C-X-C motif) ligand-9 in cardiac macrophages. Differential expression of immunomodulatory M2-like markers (eg, arginase-1, macrophage scavenger receptor 1) was dependent on the tissue location of wild-type and MR-null macrophages. Finally, intact MR signaling is required for the phosphorylation of c-Jun NH2-terminal kinase in response to a proinflammatory stimulus in bone marrow monocytes/macrophages in culture. These data suggest that the activation of the c-Jun NH2-terminal kinase pathway in macrophages after a tissue injury and inflammatory stimuli in the DOC/salt model is MR dependent and regulates the transcription of downstream profibrotic factors, which may represent potential therapeutic targets in heart failure patients.
Assuntos
Desoxicorticosterona/farmacologia , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Miocárdio/patologia , Receptores de Mineralocorticoides/agonistas , Cloreto de Sódio na Dieta/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Células Cultivadas , Fibrose , Coração/efeitos dos fármacos , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Miocárdio/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Loss of mineralocorticoid receptor signaling selectively in cardiomyocytes can ameliorate cardiac fibrotic and inflammatory responses caused by excess mineralocorticoids. The aim of this study was to characterize the role of cardiomyocyte mineralocorticoid receptor signaling in ischemia-reperfusion injury and recovery and to identify a role of mineralocorticoid receptor modulation of cardiac function. Wild-type and cardiomyocyte mineralocorticoid receptor knockout mice (8 weeks) were uninephrectomized and maintained on (1) high salt (0.9% NaCl, 0.4% KCl) or (2) high salt plus deoxycorticosterone pellet (0.3 mg/d, 0.9% NaCl, 0.4% KCl). After 8 weeks of treatment, hearts were isolated and subjected to 20 minutes of global ischemia plus 45 minutes of reperfusion. Mineralocorticoid excess increased peak contracture during ischemia regardless of genotype. Recovery of left ventricular developed pressure and rates of contraction and relaxation post ischemia-reperfusion were greater in knockout versus wild-type hearts. The incidence of arrhythmic activity during early reperfusion was significantly higher in wild-type than in knockout hearts. Levels of autophosphorylated Ca(2+)/calmodulin protein kinase II (Thr287) were elevated in hearts from wild-type versus knockout mice and associated with increased sodium hydrogen exchanger-1 expression. These findings demonstrate that cardiomyocyte-specific mineralocorticoid receptor-dependent signaling contributes to electromechanical vulnerability in acute ischemia-reperfusion via a mechanism involving Ca(2+)/calmodulin protein kinase II activation in association with upstream alteration in expression regulation of the sodium hydrogen exchanger-1.
Assuntos
Coração/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/fisiologia , Receptores de Mineralocorticoides/fisiologia , Recuperação de Função Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Animais , Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Contração Miocárdica/fisiologia , Receptores de Estrogênio/fisiologia , Receptores de Mineralocorticoides/deficiência , Receptores de Mineralocorticoides/genética , Fatores Sexuais , Trocadores de Sódio-Hidrogênio/fisiologiaRESUMO
Aldosterone plays a major role in the regulation of sodium and potassium homeostasis and blood pressure. More recently, aldosterone has emerged as a key hormone mediating end organ damage. In extreme cases, dysregulated aldosterone production leads to primary aldosteronism (PA), the most common form of secondary hypertension. However, even within the physiological range, high levels of aldosterone are associated with an increased risk of developing hypertension over time. PA represents the most common and curable form of hypertension, with a prevalence that increases with the severity of hypertension. Although genetic causes underlying glucocorticoid-remediable aldosteronism, one of the three Mendelian forms of PA, were established some time ago, somatic and inherited mutations in the potassium channel GIRK4 have only recently been implicated in the formation of aldosterone-producing adenoma (APA) and in familial hyperaldosteronism type 3. Moreover, recent findings have shown somatic mutations in two additional genes, involved in maintaining intracellular ionic homeostasis and cell membrane potential, in a subset of APAs. This review summarizes our current knowledge on the genetic determinants that contribute to variations in plasma aldosterone and renin levels in the general population and the genetics of familial and sporadic PA. Various animal models that have significantly improved our understanding of the pathophysiology of excess aldosterone production are also discussed. Finally, we outline the cardiovascular, renal, and metabolic consequences of mineralocorticoid excess beyond blood pressure regulation.
Assuntos
Aldosterona/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/complicações , Hiperaldosteronismo/genética , Hipertensão/etiologia , Renina/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Aldosterona/biossíntese , Aldosterona/sangue , Aldosterona/genética , Animais , Modelos Animais de Doenças , Humanos , Hiperaldosteronismo/sangue , Hiperaldosteronismo/metabolismo , Hipertensão/sangue , Hipertensão/genética , Mineralocorticoides/genética , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Renina/sangue , Renina/genética , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Somatic mutations of KCNJ5, coding for the potassium channel GIRK4, have recently been implicated in the formation of aldosterone producing adenoma (APA). While a causal link between KCNJ5 mutations, membrane depolarization and aldosterone production has been established, the precise mechanism by which these mutations promote cell proliferation and APA formation remains unclear. The aim of our study was to correlate KCNJ5 mutation status with morphological and functional characteristics of the adrenal cortex adjacent to APA. While GIRK4 was expressed in APA and in the zona glomerulosa of the adjacent cortex, significantly lower levels were detected in APA harboring a KCNJ5 mutation. There was no correlation between KCNJ5 mutation status and the morphological measures of adrenal cortex remodeling, including nodulation, vascularization and expression of CYP11B2. The cell composition of APA was not significantly different between groups. These results indicate that KCNJ5 mutations are not correlated with adrenal cortex remodeling in APA.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Zona Glomerulosa/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Aldosterona/biossíntese , Proliferação de Células , Citocromo P-450 CYP11B2/biossíntese , Citocromo P-450 CYP11B2/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Masculino , Mutação , RNA Mensageiro/biossínteseRESUMO
Mineralocorticoid receptor (MR) activation promotes the development of cardiac fibrosis and heart failure. Clinical evidence demonstrates that MR antagonism is protective even when plasma aldosterone levels are not increased. We hypothesize that MR activation in macrophages drives the profibrotic phenotype in the heart even when aldosterone levels are not elevated. The aim of the present study was to establish the role of macrophage MR signaling in mediating cardiac tissue remodeling caused by nitric oxide (NO) deficiency, a mineralocorticoid-independent insult. Male wild-type (MRflox/flox) and macrophage MR-knockout (MRflox/flox/LysMCre/+; mac-MRKO) mice were uninephrectomized, maintained on 0.9% NaCl drinking solution, with either vehicle (control) or the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (L-NAME; 150 mg/kg/d) for 8 wk. NO deficiency increased systolic blood pressure at 4 wk in wild-type L-NAME/salt-treated mice compared with all other groups. At 8 wk, systolic blood pressure was increased above control in both L-NAME/salt treated wild-type and mac-MRKO mice by approximately 28 mm Hg by L-NAME/salt. Recruitment of macrophages was increased 2- to 3-fold in both L-NAME/salt treated wild-type and mac-MRKO. Inducible NOS positive macrophage infiltration and TNFα mRNA expression was greater in wild-type L-NAME/salt-treated mice compared with mac-MRKO, demonstrating that loss of MR reduces M1 phenotype. mRNA levels for markers of vascular inflammation and oxidative stress (NADPH oxidase 2, p22phox, intercellular adhesion molecule-1, G protein-coupled chemokine receptor 5) were similar in treated wild-type and mac-MRKO mice compared with control groups. In contrast, L-NAME/salt treatment increased interstitial collagen deposition in wild-type by about 33% but not in mac-MRKO mice. mRNA levels for connective tissue growth factor and collagen III were also increased above control treatment in wild-type (1.931 ± 0.215 vs. 1 ± 0.073) but not mac-MRKO mice (1.403 ± 0.150 vs. 1.286 ± 0.255). These data demonstrate that macrophage MR are necessary for the translation of inflammation and oxidative stress into interstitial and perivascular fibrosis after NO deficiency, even when plasma aldosterone is not elevated.
Assuntos
Aldosterona/metabolismo , Fibrose/metabolismo , Coração/fisiologia , Macrófagos/citologia , Receptores de Mineralocorticoides/metabolismo , Angiotensina Amida/metabolismo , Animais , Hipertensão/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Fenótipo , Transdução de Sinais , Fatores de TempoAssuntos
Aldosterona/farmacologia , Aorta/efeitos dos fármacos , Receptores ErbB/fisiologia , Vasoconstrição/efeitos dos fármacos , Acetilcolina/farmacologia , Angiotensina II/farmacologia , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Hemodinâmica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Mutantes , Modelos Biológicos , Receptores de Mineralocorticoides/fisiologia , Transdução de Sinais/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Increased mineralocorticoid levels plus high salt promote vascular inflammation and cardiac tissue remodeling. Mineralocorticoid receptors are expressed in many cell types of the cardiovascular system, including monocytes/macrophages and other inflammatory cell types. Although mineralocorticoid receptors are expressed in monocytes/macrophages, their role in regulating macrophage function to date has not been investigated. We, thus, used the Cre/LoxP-recombination system to selectively delete mineralocorticoid receptors from monocytes/macrophages with the lysozyme M promoter used to drive Cre expression (MR(flox/flox)/LysM(Cre/-) mice). Male mice from each genotype (MR(flox/flox) or wild-type and MR(flox/flox)/LysM(Cre/-) mice) were uninephrectomized, given 0.9% NaCl solution to drink, and treated for 8 days or 8 weeks with either vehicle (n=10) or deoxycorticosterone (n=10). Equivalent tissue macrophage numbers were seen for deoxycorticosterone treatment of each genotype at 8 days; in contrast, plasminogen activator inhibitor type 1 and NAD(P)H oxidase subunit 2 levels were increased in wild-type but not in MR(flox/flox)/LysM(Cre/-) mice given deoxycorticosterone. Baseline expression of other inflammatory genes was reduced in MR(flox/flox)/LysM(Cre/-) mice compared with wild-type mice. At 8 weeks, deoxycorticosterone-induced macrophage recruitment and connective tissue growth factor and plasminogen activator inhibitor type 1 mRNA levels were similar for each genotype; in contrast, MR(flox/flox)/LysM(Cre/-) mice showed no increase in cardiac fibrosis or blood pressure, as was seen in wild-type mice at 8 weeks. These data demonstrate the following points: (1) mineralocorticoid receptor signaling regulates basal monocyte/macrophage function; (2) macrophage recruitment is not altered by loss of mineralocorticoid receptor signaling in these cells; and (3) a novel and significant role is seen for macrophage signaling in the regulation of cardiac remodeling and systolic blood pressure in the deoxycorticosterone/salt model.
Assuntos
Pressão Sanguínea/fisiologia , Macrófagos/metabolismo , Miocárdio/patologia , Receptores de Mineralocorticoides/fisiologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Pressão Sanguínea/efeitos dos fármacos , Quimiocina CCL2/genética , Colágeno/metabolismo , Desoxicorticosterona/administração & dosagem , Desoxicorticosterona/efeitos adversos , Feminino , Fibrose/etiologia , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Miocárdio/metabolismo , Nefrectomia , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptores de Mineralocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio na Dieta/administração & dosagem , Cloreto de Sódio na Dieta/efeitos adversosRESUMO
The mineralocorticoid receptor (MR) and glucocorticoid receptor are ligand-activated transcription factors that have important physiological and pathophysiological actions in a broad range of cell types including monocytes and macrophages. While the glucocorticoids cortisol and corticosterone have well-described anti-inflammatory actions on both recruited and tissue resident macrophages, a role for the mineralocorticoid aldosterone in these cells is largely undefined. Emerging evidence, however, suggests that MR signalling may promote pro-inflammatory effects. This review will discuss the current understanding of the role of corticosteroid receptors in macrophages and their effect on diseases involving inflammation, with a particular focus on cardiovascular disease.